Funded by this grant, my lab has combined biochemistry and cell biology to define the role of the GTPase dynamin in endocytic clathrin-coated vesicle (CCV) formation. Our data, and results of others, now suggest that dynamin plays a dual role in clathrin-mediated endocytosis (CME). During early stages of CME, unassembled dynamin is localized to coated pits and functions as a regulatory GTPase to monitor coat assembly, cargo capture and membrane curvature. At late stages, dynamin self-assembles around the neck of deeply invaginated coated pits and rapid, assembly-stimulated GTP hydrolysis drives a concerted conformational change necessary for membrane fission. Thus, a consensus has emerged that dynamin is a mechanochemical enzyme, but little is known about the molecular nature of dynamin's mechanochemical conformational changes that govern dynamin-mediated membrane fission. Over the next 4 years we will rigorously apply biochemical, biophysical and structural approaches to define the structural and enzymological basis for dynamin's mechanochemical properties and to identify how the enzymatic and mechanochemical activities of dynamin function together with coat proteins and other endocytic accessory factors to mediate CCV formation. In Aim 1 we will characterize new classes of dynamin mutants that differentially affect basal and assembly- stimulated GTPase activity and test the functions of these distinct dynamin activities in CCV formation in vivo by reconstitution of conditional dynamin-2 knock-out cells. In Aim 2, we will use X-ray crystallography and cryo-EM, together with helical reconstruction techniques to define the structural bases for basal and assembly-stimulated GTPase activities. In Aim 3 we will use site- specific labeling of dynamin and multiple independent fluorescence spectroscopy techniques to define the nucleotide-dependent, and effector-regulated conformational changes in dynamin that account for its mechanochemical properties towards lipid bilayers. Finally, in Aim 4 we will develop and utilize new fluorescence-based assays to directly measure dynamin-dependent steps in CCV formation from supported proteolipid bilayers. Together these studies should provide a detailed mechanistic understanding of dynamin function in membrane fission. As dynamin is a prototypical member of a diverse class a GTPases involved in membrane fission, the results from our studies will have broad impact on many areas of cell biology.